Sulfated Hydrogels as Primary Intervertebral Disc Cell Culture Systems.

The negatively charged extracellular matrix plays a vital role in intervertebral disc tissues, providing specific cues for cell maintenance and tissue hydration. Unfortunately, suitable biomimetics for intervertebral disc regeneration are lacking. Here, sulfated alginate was investigated as a 3D culture material due to its similarity to the charged matrix of the intervertebral disc. Precursor solutions of standard alginate, or alginate with 0.1% or 0.2% degrees of sulfation, were mixed with primary human nucleus pulposus cells, cast, and cultured for 14 days. A 0.2% degree of sulfation resulted in significantly decreased cell density and viability after 7 days of culture. Furthermore, a sulfation-dependent decrease in DNA content and metabolic activity was evident after 14 days. Interestingly, no significant differences in cell density and viability were observed between surface and core regions for sulfated alginate, unlike in standard alginate, where the cell number was significantly higher in the core than in the surface region. Due to low cell numbers, phenotypic evaluation was not achieved in sulfated alginate biomaterial. Overall, standard alginate supported human NP cell growth and viability superior to sulfated alginate; however, future research on phenotypic properties is required to decipher the biological properties of sulfated alginate in intervertebral disc cells.

The influence of physical and spatial substrate characteristics on endothelial cells.

Cardiovascular diseases are a main cause of death worldwide, leading to a growing demand for medical devices to treat this patient group. Central to the engineering of such devices is a good understanding of the biology and physics of cell-surface interactions. In existing blood-contacting devices, such as vascular grafts, the interaction between blood, cells, and material is one of the main limiting factors for their long-term durability. An improved understanding of the material's chemical- and physical properties as well as its structure all play a role in how endothelial cells interact with the material surface. This review provides an overview of how different surface structures influence endothelial cell responses and what is currently known about the underlying mechanisms that guide this behavior. The structures reviewed include decellularized matrices, electrospun fibers, pillars, pits, and grated surfaces.

Exploring the Impact of TLR-2 Signaling on miRNA Dysregulation in Intervertebral Disc Degeneration.

Toll-like receptors (TLRs) are key mediators of inflammation in intervertebral disc (IVD) degeneration. TLR-2 activation contributes to the degenerative process by increasing the expression of extracellular matrix-degrading enzymes, pro-inflammatory cytokines, and neurotrophins. As potent post-transcriptional regulators, microRNAs can modulate intracellular mechanisms, and their dysregulation is known to contribute to numerous pathologies. This study aims to investigate the impact of TLR-2 signaling on miRNA dysregulation in the context of IVD degeneration. Small-RNA sequencing of degenerated IVD cells shows the dysregulation of ten miRNAs following TLR-2 activation by PAM2CSK4. The miR-155-5p is most significantly upregulated in degenerated and non-degenerated annulus fibrosus and nucleus pulposus cells. Sequence-based target and pathway prediction shows the involvement of miR-155-5p in inflammation- and cell fate-related pathways and TLR-2-induced miR-155-5p expression leads to the downregulation of its target c-FOS. Furthermore, changes specific to the activation of TLR-2 through fragmented fibronectin are seen in miR-484 and miR-487. Lastly, miR-100-3p, miR-320b, and miR-181a-3p expression exhibit degeneration-dependent changes. These results show that TLR-2 signaling leads to the dysregulation of miRNAs in IVD cells as well as their possible downstream effects on inflammation and degeneration. The identified miRNAs provide important opportunities as potential therapeutic targets for IVD degeneration and low back pain.

Aloe vera-based biomaterial ink for 3D bioprinting of wound dressing constructs.

This study emphasizes the development of a multifunctional biomaterial ink for wound healing constructs. The biomaterial ink benefits from Aloe vera's intrinsic biocompatible, biodegradable, antioxidant, antimicrobial, anti-inflammatory, and immunomodulatory attributes, thus alleviating the need for supplementary substances employed to combat infections and stimulate tissue regeneration. Moreover, this biomaterial ink seeks to address the scarcity of standardized printable materials possessing adequate biocompatibility and physicochemical properties, which hinder its widespread clinical adoption. The biomaterial ink was synthesized via ionic crosslinking to enhance its rheological and mechanical characteristics. The findings revealed that Aloe vera substantially boosted the hydrogel's viscoelastic behavior, enabling superior compressive modulus and the extrusion of fine filaments. The bioprinted constructs exhibited desirable resolution and mechanical strength while displaying a porous microstructure analogous to the native extracellular matrix. Biological response demonstrated no detrimental impact on stem cell viability upon exposure to the biomaterial ink, as confirmed by live/dead assays. These outcomes validate the potential of the developed biomaterial ink as a resource for the bioprinting of wound dressings that effectively foster cellular proliferation, thereby promoting enhanced wound healing by leveraging Aloe vera's inherent properties.

Targeted screening of inflammatory mediators in spontaneous degenerative disc disease in dogs reveals an upregulation of the tumor necrosis superfamily.

Background: The regulation of inflammatory mediators in the degenerating intervertebral disc (IVD) and corresponding ligamentum flavum (LF) is a topic of emerging interest. The study aimed to investigate the expression of a broad array of inflammatory mediators in the degenerated LF and IVD using a dog model of spontaneous degenerative disc disease (DDD) to determine potential treatment targets. Methods: LF and IVD tissues were collected from 22 normal dogs (Pfirrmann grades I and II) and 18 dogs affected by DDD (Pfirrmann grades III and IV). A qPCR gene array was used to investigate the expression of 80 inflammatory genes for LF and IVD tissues, whereafter targets of interest were investigated in additional tissue samples using qPCR, western blot (WB), and immunohistochemistry. Results: Tumor necrosis factor superfamily (TNFSF) signaling was identified as a regulated pathway in DDD, based on the significant regulation (n-fold ± SD) of various TNFSF members in the degenerated IVD, including nerve growth factor (NGF; -8 ± 10), CD40LG (464 ± 442), CD70 (341 ± 336), TNFSF Ligand 10 (9 ± 8), and RANKL/TNFSF Ligand 11 (85 ± 74). In contrast, TNFSF genes were not significantly affected in the degenerated LF compared to the control LF. Protein expression of NGF (WB) was significantly upregulated in both the degenerated LF (4.4 ± 0.5) and IVD (11.3 ± 5.6) compared to the control group. RANKL immunopositivity was significantly upregulated in advanced stages of degeneration (Thompson grades IV and V) in the nucleus pulposus and annulus fibrosus of the IVD, but not in the LF. Conclusions: DDD involves a significant upregulation of various TNFSF members, with tissue-specific expression profiles in LF and IVD tissues. The differential involvement of TNFSF members within multiple spinal tissues from the same individual provides new insights into the inflammatory processes involved in DDD and may provide a basis to formulate hypotheses for the determination of potential treatment targets.

Ibuprofen-loaded electrospun poly(ethylene-co-vinyl alcohol) nanofibers for wound dressing applications.

Chronic wounds are characterized by a prolonged inflammation phase preventing the normal processes of wound healing and natural regeneration of the skin. To tackle this issue, electrospun nanofibers, inherently possessing a high surface-to-volume ratio and high porosity, are promising candidates for the design of anti-inflammatory drug delivery systems. In this study, we evaluated the ability of poly(ethylene-co-vinyl alcohol) nanofibers of various chemical compositions to release ibuprofen for the potential treatment of chronic wounds. First, the electrospinning of poly(ethylene-co-vinyl alcohol) copolymers with different ethylene contents (32, 38 and 44 mol%) was optimized in DMSO. The morphology and surface properties of the membranes were investigated via state-of-the-art techniques and the influence of the ethylene content on the mechanical and thermal properties of each membrane was evaluated. Furthermore, the release kinetics of ibuprofen from the nanofibers in a physiological temperature range revealed that more ibuprofen was released at 37.5 °C than at 25 °C regardless of the ethylene content. Additionally, at 25 °C less drug was released when the ethylene content of the membranes increased. Finally, the scaffolds showed no cytotoxicity to normal human fibroblasts collectively paving the way for the design of electrospun based patches for the treatment of chronic wounds.

Clinical and radiological mid- to long-term investigation of anterior lumbar stand-alone fusion: Incidence of reoperation and adjacent segment degeneration.

Introduction: Anterior stand-alone fusion (ASAF) devices have been developed in an attempt to reduce adjacent segment degenerative changes observed with posterior instrumented fusion techniques. Research question: The purpose of this study was to assess mid- to long-term clinical and radiological results following ASAF at the lumbosacral junction with special emphasis on the assessment of adjacent level pathologies.Materials & Methods: Clinical outcome scores and radiological data were acquired within an ongoing single-center prospective cohort study. Progression of adjacent level degeneration was evaluated based on MRI scans according to the Pfirrmann and Weishaupt classification system by two independent radiologists. Results: The results from 37 patients (FU â€‹≥ â€‹5 years) demonstrated high satisfaction rates and significant improvements in VAS and ODI scores. N â€‹= â€‹8 patients (21.6%) had to undergo subsequent surgery at the cranially adjacent level. The incidence of adjacent level disc degeneration and adjacent facet joint degeneration was 24.3% and 35.1%, respectively. More pronounced degenerative changes of the adjacent level discs (p â€‹= â€‹0.005) and facet joints (p â€‹= â€‹0.042) prior to surgery and a lower segmental lordosis reconstruction at the lumbosacral junction (p â€‹= â€‹0.0084) were identified as potential risk factors for the development of subsequent adjacent level pathologies. Discussion & Conclusion: The study revealed satisfactory clinical results at a mid-to long-term FU of ≥5 years. The incidence of adjacent level degeneration was higher than initially expected. Patients with preexisting radiographic signs of degenerative adjacent level changes have a higher risk for subsequent deterioration necessitating reoperation at the adjacent segment at later stages. Furthermore, adequate intraoperative segmental lordosis reconstruction at the index segment is paramount as the present data reveal this to be a key protective factor for adjacent segment preservation.

Immuno-Modulatory Effects of Intervertebral Disc Cells.

Low back pain is a highly prevalent, chronic, and costly medical condition predominantly triggered by intervertebral disc degeneration (IDD). IDD is often caused by structural and biochemical changes in intervertebral discs (IVD) that prompt a pathologic shift from an anabolic to catabolic state, affecting extracellular matrix (ECM) production, enzyme generation, cytokine and chemokine production, neurotrophic and angiogenic factor production. The IVD is an immune-privileged organ. However, during degeneration immune cells and inflammatory factors can infiltrate through defects in the cartilage endplate and annulus fibrosus fissures, further accelerating the catabolic environment. Remarkably, though, catabolic ECM disruption also occurs in the absence of immune cell infiltration, largely due to native disc cell production of catabolic enzymes and cytokines. An unbalanced metabolism could be induced by many different factors, including a harsh microenvironment, biomechanical cues, genetics, and infection. The complex, multifactorial nature of IDD brings the challenge of identifying key factors which initiate the degenerative cascade, eventually leading to back pain. These factors are often investigated through methods including animal models, 3D cell culture, bioreactors, and computational models. However, the crosstalk between the IVD, immune system, and shifted metabolism is frequently misconstrued, often with the assumption that the presence of cytokines and chemokines is synonymous to inflammation or an immune response, which is not true for the intact disc. Therefore, this review will tackle immunomodulatory and IVD cell roles in IDD, clarifying the differences between cellular involvements and implications for therapeutic development and assessing models used to explore inflammatory or catabolic IVD environments.

Resveratrol Microencapsulation into Electrosprayed Polymeric Carriers for the Treatment of Chronic, Non-Healing Wounds.

Chronic, non-healing wounds represent a challenging socio-economic burden, demanding innovative approaches for successful wound management. Resveratrol (RSV) represents a promising therapeutic candidate, but its therapeutic efficacy and clinical applicability have been hampered by its rapid degradation and/or depletion. Herein, RSV was encapsulated into poly(ε-caprolactone) (PCL) microparticles by electrospraying with the aim to prolong and preserve RSV's release/activity, without affecting its therapeutic properties. Electrospraying led to the fabrication of spherical (2 to 10 µm in size), negatively charged (<−1 mV), and quasi-monodisperse (PDI < 0.3) microparticles, with 60% RSV release after 28 days. Microencapsulation of RSV into PCL prevented its photochemical degradation and preserved its antioxidant properties over 72 h. The RSV-PCL microparticles did not exhibit any cytotoxicity on human dermal fibroblasts. RSV released from the microparticles was biologically functional and induced a significant increase in collagen type I deposition. Furthermore, the produced RSV-PCL microparticles reduced the expression of inflammatory (IL-6, IL-8, COX-2) and proteolytic (MMP-2, MMP-9) mediators. Collectively, our data clearly illustrate the potential of electrosprayed polymeric carriers for the sustained delivery of RSV to treat chronic wounds.

Acrylonitrile and Pullulan Based Nanofiber Mats as Easily Accessible Scaffolds for 3D Skin Cell Models Containing Primary Cells.

(1) Background: Three-dimensional (3D) collagen I-based skin models are commonly used in drug development and substance testing but have major drawbacks such as batch-to-batch variations and ethical concerns. Recently, synthetic nanofibrous scaffolds created by electrospinning have received increasing interest as potential alternatives due to their morphological similarities to native collagen fibrils in size and orientation. The overall objective of this proof-of-concept study was to demonstrate the suitability of two synthetic polymers in creating electrospun scaffolds for 3D skin cell models. (2) Methods: Electrospun nanofiber mats were produced with (i) poly(acrylonitrile-co-methyl acrylate) (P(AN-MA)) and (ii) a blend of pullulan (Pul), poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) (Pul/PVA/PAA) and characterized by scanning electron microscopy (SEM) and diffuse reflectance infrared Fourier transform (DRIFT) spectra. Primary skin fibroblasts and keratinocytes were seeded onto the nanofiber mats and analyzed for phenotypic characteristics (phalloidin staining), viability (Presto Blue HS assay), proliferation (Ki-67 staining), distribution (H/E staining), responsiveness to biological stimuli (qPCR), and formation of skin-like structures (H/E staining). (3) Results: P(AN-MA) mats were more loosely packed than the Pul/PVA/PAA mats, concomitant with larger fiber diameter (340 nm ± 120 nm vs. 250 nm ± 120 nm, p < 0.0001). After sterilization and exposure to cell culture media for 28 days, P(AN-MA) mats showed significant adsorption of fetal calf serum (FCS) from the media into the fibers (DRIFT spectra) and increased fiber diameter (590 nm ± 290 nm, p < 0.0001). Skin fibroblasts were viable over time on both nanofiber mats, but suitable cell infiltration only occurred in the P(AN-MA) nanofiber mats. On P(AN-MA) mats, fibroblasts showed their characteristic spindle-like shape, produced a dermis-like structure, and responded well to TGFß stimulation, with a significant increase in the mRNA expression of PAI1, COL1A1, and αSMA (all p < 0.05). Primary keratinocytes seeded on top of the dermis equivalent proliferated and formed a stratified epidermis-like structure. (4) Conclusion: P(AN-MA) and Pul/PVA/PAA are both biocompatible materials suitable for nanofiber mat production. P(AN-MA) mats hold greater potential as future 3D skin models due to enhanced cell compatibility (i.e., adsorption of FCS proteins), cell infiltration (i.e., increased pore size due to swelling behavior), and cell phenotype preservation. Thus, our proof-of-concept study shows an easy and robust process of producing electrospun scaffolds for 3D skin cell models made of P(AN-MA) nanofibers without the need for bioactive molecule attachments.

Sulfated Hydrogels in Intervertebral Disc and Cartilage Research.

Hydrogels are commonly used for the 3D culture of musculoskeletal cells. Sulfated hydrogels, which have seen a growing interest over the past years, provide a microenvironment that help maintain the phenotype of chondrocytes and chondrocyte-like cells and can be used for sustained delivery of growth factors and other drugs. Sulfated hydrogels are hence valuable tools to improve cartilage and intervertebral disc tissue engineering. To further advance the utilization of these hydrogels, we identify and summarize the current knowledge about different sulfated hydrogels, highlight their beneficial effects in cartilage and disc research, and review the biofabrication processes most suitable to secure best quality assurance through deposition fidelity, repeatability, and attainment of biocompatible morphologies.

TRPV4 mediates cell damage induced by hyperphysiological compression and regulates COX2/PGE2 in intervertebral discs.

BACKGROUND: Aberrant mechanical loading of the spine causes intervertebral disc (IVD) degeneration and low back pain. Current therapies do not target the mediators of the underlying mechanosensing and mechanotransduction pathways, as these are poorly understood. This study investigated the role of the mechanosensitive transient receptor potential vanilloid 4 (TRPV4) ion channel in dynamic compression of bovine nucleus pulposus (NP) cells in vitro and mouse IVDs in vivo. METHODS: Degenerative changes and the expression of the inflammatory mediator cyclooxygenase 2 (COX2) were examined histologically in the IVDs of mouse tails that were dynamically compressed at a short repetitive hyperphysiological regime (vs sham). Bovine NP cells embedded in an agarose-collagen hydrogel were dynamically compressed at a hyperphysiological regime in the presence or absence of the selective TRPV4 antagonist GSK2193874. Lactate dehydrogenase (LDH) and prostaglandin E2 (PGE2) release, as well as phosphorylation of mitogen-activated protein kinases (MAPKs), were analyzed. Degenerative changes and COX2 expression were further evaluated in the IVDs of trpv4-deficient mice (vs wild-type; WT). RESULTS: Dynamic compression caused IVD degeneration in vivo as previously shown but did not affect COX2 expression. Dynamic compression significantly augmented LDH and PGE2 releases in vitro, which were significantly reduced by TRPV4 inhibition. Moreover, TRPV4 inhibition during dynamic compression increased the activation of the extracellular signal-regulated kinases 1/2 (ERK) MAPK pathway by 3.13-fold compared to non-compressed samples. Trpv4-deficient mice displayed mild IVD degeneration and decreased COX2 expression compared to WT mice. CONCLUSIONS: TRPV4 therefore regulates COX2/PGE2 and mediates cell damage induced by hyperphysiological dynamic compression, possibly via ERK. Targeted TRPV4 inhibition or knockdown might thus constitute promising therapeutic approaches to treat patients suffering from IVD pathologies caused by aberrant mechanical stress.

pH-Responsive Chitosan/Alginate Polyelectrolyte Complexes on Electrospun PLGA Nanofibers for Controlled Drug Release.

The surface functionalization of electrospun nanofibers allows for the introduction of additional functionalities while at the same time retaining the membrane properties of high porosity and surface-to-volume ratio. In this work, we sequentially deposited layers of chitosan and alginate to form a polyelectrolyte complex via layer-by-layer assembly on PLGA nanofibers to introduce pH-responsiveness for the controlled release of ibuprofen. The deposition of the polysaccharides on the surface of the fibers was revealed using spectroscopy techniques and ζ-potential measurements. The presence of polycationic chitosan resulted in a positive surface charge (16.2 ± 4.2 mV, pH 3.0) directly regulating the interactions between a model drug (ibuprofen) loaded within the polyelectrolyte complex and the layer-by-layer coating. The release of ibuprofen was slowed down in acidic pH (1.0) compared to neutral pH as a result of the interactions between the drug and the coating. The provided mesh acts as a promising candidate for the design of drug delivery systems required to bypass the acidic environment of the digestive tract.

Extracellular Vesicles: Potential Mediators of Psychosocial Stress Contribution to Osteoporosis?

Osteoporosis is characterized by low bone mass and damage to the bone tissue's microarchitecture, leading to increased fracture risk. Several studies have provided evidence for associations between psychosocial stress and osteoporosis through various pathways, including the hypothalamic-pituitary-adrenocortical axis, the sympathetic nervous system, and other endocrine factors. As psychosocial stress provokes oxidative cellular stress with consequences for mitochondrial function and cell signaling (e.g., gene expression, inflammation), it is of interest whether extracellular vesicles (EVs) may be a relevant biomarker in this context or act by transporting substances. EVs are intercellular communicators, transfer substances encapsulated in them, modify the phenotype and function of target cells, mediate cell-cell communication, and, therefore, have critical applications in disease progression and clinical diagnosis and therapy. This review summarizes the characteristics of EVs, their role in stress and osteoporosis, and their benefit as biological markers. We demonstrate that EVs are potential mediators of psychosocial stress and osteoporosis and may be beneficial in innovative research settings.

Engineering Advanced In Vitro Models of Systemic Sclerosis for Drug Discovery and Development.

Systemic sclerosis (SSc) is a complex multisystem disease with the highest case-specific mortality among all autoimmune rheumatic diseases, yet without any available curative therapy. Therefore, the development of novel therapeutic antifibrotic strategies that effectively decrease skin and organ fibrosis is needed. Existing animal models are cost-intensive, laborious and do not recapitulate the full spectrum of the disease and thus commonly fail to predict human efficacy. Advanced in vitro models, which closely mimic critical aspects of the pathology, have emerged as valuable platforms to investigate novel pharmaceutical therapies for the treatment of SSc. This review focuses on recent advancements in the development of SSc in vitro models, sheds light onto biological (e.g., growth factors, cytokines, coculture systems), biochemical (e.g., hypoxia, reactive oxygen species) and biophysical (e.g., stiffness, topography, dimensionality) cues that have been utilized for the in vitro recapitulation of the SSc microenvironment, and highlights future perspectives for effective drug discovery and validation.

Effect of BMI on the clinical outcome following microsurgical decompression in over-the-top technique: bi-centric study with an analysis of 744 patients.

PURPOSE: Decompression is one of the most common interventions in spinal surgery. Obesity has become an increasing issue in surgical patients. Therefore, the aim of this study was to analyze the clinical outcome following lumbar microsurgical decompression in correlation with the patient's body mass index (BMI). MATERIALS AND METHODS: A toal of 744 patients with spinal claudication that were seen at two specialized spine centers were included in this study. All patients underwent a bilateral microsurgical decompression in over-the-top technique. Patients were allocated in 4 groups based in their BMI category: 18.5-24.9 (n = 204), 25.0-29.9 (n = 318), 30.0-34.9 (n = 164) and ≥ 35 (n = 58). Clinical outcome data were recorded at baseline as well as 3, 12 and 24 months thereafter within a prospective study framework. The minimum follow-up was 12 months. For statistical analysis, data were adjusted for age, length of surgery and ASA and were analyzed by generalized linear gamma-based models. RESULTS: Postoperative changes in all outcome parameters were clearly dependent on BMI. Patients with higher BMI were characterized by inferior baseline values for VAS Back (p < 0.05). Over the follow-up period, the groups with BMI 30-34.9 and BMI ≥ 35 benefitted significantly less from the surgery than the two slimmer groups (p < 0.05). CONCLUSION: The data obtained from this large, homogenous cohort demonstrate that the quality of life improved substantially in all patients that were treated microsurgically for spinal stenosis. However, a BMI ≥ 30 may be considered as a negative predictor for a significantly inferior clinical outcome.

Multiscale Regulation of the Intervertebral Disc: Achievements in Experimental, In Silico, and Regenerative Research.

Intervertebral disc (IVD) degeneration is a major risk factor of low back pain. It is defined by a progressive loss of the IVD structure and functionality, leading to severe impairments with restricted treatment options due to the highly demanding mechanical exposure of the IVD. Degenerative changes in the IVD usually increase with age but at an accelerated rate in some individuals. To understand the initiation and progression of this disease, it is crucial to identify key top-down and bottom-up regulations' processes, across the cell, tissue, and organ levels, in health and disease. Owing to unremitting investigation of experimental research, the comprehension of detailed cell signaling pathways and their effect on matrix turnover significantly rose. Likewise, in silico research substantially contributed to a holistic understanding of spatiotemporal effects and complex, multifactorial interactions within the IVD. Together with important achievements in the research of biomaterials, manifold promising approaches for regenerative treatment options were presented over the last years. This review provides an integrative analysis of the current knowledge about (1) the multiscale function and regulation of the IVD in health and disease, (2) the possible regenerative strategies, and (3) the in silico models that shall eventually support the development of advanced therapies.

Uncovering the secretome of mesenchymal stromal cells exposed to healthy, traumatic, and degenerative intervertebral discs: a proteomic analysis.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been introduced as promising cell source for regenerative medicine. Besides their multilineage differentiation capacity, MSCs release a wide spectrum of bioactive factors. This secretome holds immunomodulatory and regenerative capacities. In intervertebral disc (IVD) cells, application of MSC secretome has been shown to decrease the apoptosis rate, induce proliferation, and promote production of extracellular matrix (ECM). For clinical translation of secretome-based treatment, characterization of the secretome composition is needed to better understand the induced biological processes and identify potentially effective secretomes. METHODS: This study aimed to investigate the proteome released by bone marrow-derived MSCs following exposure to a healthy, traumatic, or degenerative human IVD environment by mass spectroscopy and quantitative immunoassay analyses. Exposure of MSCs to the proinflammatory stimulus interleukin 1ß (IL-1ß) was used as control. RESULTS: Compared to MSC baseline secretome, there were 224 significantly up- or downregulated proteins following healthy, 179 following traumatic, 223 following degenerative IVD, and 160 proteins following IL-1ß stimulus. Stimulation of MSCs with IVD conditioned media induced a more complex MSC secretome, involving more biological processes, compared to stimulation with IL-1ß. The MSC response to stimulation with IVD conditioned medium was dependent on their pathological status. CONCLUSIONS: The MSC secretome seemed to match the primary need of the IVD: homeostasis maintenance in the case of healthy IVDs, versus immunomodulation, adjustment of ECM synthesis and degradation disbalance, and ECM (re) organization in the case of traumatic and degenerative IVDs. These findings highlight the importance of cell preconditioning in the development of tailored secretome therapies. The secretome of human bone marrow-derived mesenchymal stromal cells (MSCs) stimulated with intervertebral disc (IVD) conditioned medium was analyzed by proteomic profiling. Depending on the pathological state of the IVD, the MSC secretome protein composition indicated immunomodulatory or anabolic activity of the secretome. These findings may have implications for tailored secretome therapy for the IVD and other tissues.

Fibronectin Fragments and Inflammation During Canine Intervertebral Disc Disease.

Background: Canine intervertebral disc disease (IVDD) represents a significant clinical problem in veterinary medicine, with similarities to the human pathology. Host-derived damage-associated molecular patterns like fibronectin fragments (FnF) that develop during tissue dysfunction may be of specific relevance to IVD pathologies by inducing an inflammatory response in resident cells. Aim: This project aimed to determine the presence and pathobiological role of FnF during IVD herniation in dogs, with a focus on inflammation. Methods: Herniated nucleus pulposus (NP) material from five dogs as well as non-herniated adjacent NP material from three dogs was collected during spinal surgery required due to acute IVD herniation. The presence of different types of FnF were determined by Western blot analysis. NP cells isolated from six herniated canine IVDs were then exposed to 30 kDa FnF. NP cell inflammation and catabolism was examined by investigating the expression of IL-1ß, IL-6, IL-8, and COX-2, as well as MMP-1 and MMP-3 by qPCR (all targets) and ELISA (IL-6, PGE2). Results: Amongst multiple sized FnF (30, 35, 45, and >170kDa), N-terminal fragments at a size of ~30 kDa were most consistently expressed in all five herniated IVDs. Importantly, these fragments were exclusively present in herniated, but not in non-herniated IVDs. Exposure of canine NP cells to 500 nM 30 kDa FnF caused a significant upregulation of IL-6 (62.5 ± 79.9, p = 0.032) and IL-8 (53.0 ± 75.7, p = 0.031) on the gene level, whereas IL-6 protein analysis was inconclusive. Donor-donor variation was observed in response to FnF treatment, whereby this phenomenon was most evident for COX-2, with three donors demonstrating a significant downregulation (0.67 ± 0.03, p = 0.003) and three donors showing upregulation (6.9 ± 5.5, p = 0.21). Co-treatment with Sparstolonin B, a TRL-2/TRL-4 antagonist, showed no statistical difference to FnF treatment alone in all tested target genes. Conclusion: Given the presence of the 30 kDa FnF in canine herniated IVDs and the proinflammatory effect of 30 kDa FnF on NP cells, we concluded that the accumulation of FnF may be involved in the pathogenesis of canine IVDD. These results correspond to the findings in humans with IVDD.

Effects of Early Life Stress on Bone Homeostasis in Mice and Humans.

Bone pathology is frequent in stressed individuals. A comprehensive examination of mechanisms linking life stress, depression and disturbed bone homeostasis is missing. In this translational study, mice exposed to early life stress (MSUS) were examined for bone microarchitecture (µCT), metabolism (qPCR/ELISA), and neuronal stress mediator expression (qPCR) and compared with a sample of depressive patients with or without early life stress by analyzing bone mineral density (BMD) (DXA) and metabolic changes in serum (osteocalcin, PINP, CTX-I). MSUS mice showed a significant decrease in NGF, NPYR1, VIPR1 and TACR1 expression, higher innervation density in bone, and increased serum levels of CTX-I, suggesting a milieu in favor of catabolic bone turnover. MSUS mice had a significantly lower body weight compared to control mice, and this caused minor effects on bone microarchitecture. Depressive patients with experiences of childhood neglect also showed a catabolic pattern. A significant reduction in BMD was observed in depressive patients with childhood abuse and stressful life events during childhood. Therefore, future studies on prevention and treatment strategies for both mental and bone disease should consider early life stress as a risk factor for bone pathologies.